The invention has as its object a solution for perfusion and/or preservation and/or re-perfusion during organ transplant, especially of the heart.
The preservation period of human hearts is at present 4 hours and a certain number of rejections are still due to deterioration in the condition of the transplanted organ between the time of removal and the moment of implantation in the recipient.
Short-term myocardiac preservation (4 hours) is currently provided by cold storage after cardioplegic arrest. A variety of processes exist however differing by the composition of the solution used, the preservation temperature and the administration protocol. Different solutions for arresting and preserving the heart have been developed to protect the myocardium in cardiac surgery.
The St Thomas solution [Ledingham, S. J. M., Braimbridge, M. V., Hearse, D. J. The St Thomas"" Hospital cardioplegic solution: a comparison of the efficacy of two formulations. J. Thorac. Cardiovasc. Surg. (1987) 93: 240-246] has in particular been much used. 
Solutions similar to the St Thomas solution have been developed like the Broussais solution [Fabiani, J. N., Ponzio, O., Jebara, V. Myocardiac protection; encycl. Mxc3xa9d. Chir. (Paris, France), Techniques chirurgicales, Thorax, 42511, 10-1989].
More recently experiments have been carried out with the University of Wisconsin (UW) solution [Ledingham, S. J . M., Katayama, O., Lachno, D. R., Yacoub, M. H. Prolonged cardiac preservation. Evaluation of the University of Wisconsin preservation solution by comparison with the St Thomas"" solution in the rat. Circulation (1990) 82 (Part 2): IV351-8] currently used for other organs (liver, kidney). The use of the UW solution has allowed impressive progress in the preservation of the transplanted organ in renal and hepatic transplantation by significantly increasing the duration of cold ischemia before transplantation.
Certain teams have already reported favourable results by applying the UW solution to the preservation of the cardiac transplant. This solution comprises different protective elements; glutathion and allopurinol, inhibitors of the formation of free radicals from oxygen, water resistant agents (lactobionate and raffinose) and adenosine, a precursor of ATP.
Different solutions directly derived from the UW solution have been developed, yet others take only certain elements of the UW solution (Celsior solution) [Mxc3xa9naschxc3xa9, P., Termignon, J L., Pradier, F., Grousset, C., Mouas, C. Experimental evaluation of Celsior, a new heart preservation solution. Eur J. Cardio. Thorac. Surg. (1994) 8: 207-213].
It has been shown that the University of Wisconsin solution (UW) made it possible to increase the duration of myocardium preservation. This solution comprises different protective elements, glutathion and allopurinol, inhibitors of the formation of free radicals from oxygen, water resistant agents, (lactobionate, raffinose), a precursor of ATP (adenosine). However, this solution has not been specifically developed for the heart and presents various features which are not optimal if not harmful for the heart: 1) the UW solution is characterised for example by a strong concentration in K+ (125 mM) damaging to the cells as it leads to contracture, 2) inorganic phosphate (Pi) is equally present in a strong concentration or it is an inhibitor of numerous ATPases (Na+/K+ ATPase, Ca2+ ATPases) and can thus increase the accumulation in poisonous Na+ and Ca2+ in the course of ischemia, 3) it does not comprise any Ca2+ and because of this induces a massive influx of Ca2+ during re-perfusion, 4) adenosine has numerous effects on the heart and its concentration is not optimised in the solution, 5) the UW solution does not include a certain number of elements whose protective action has been able to be shown in the heart.
Several years ago a cardioplegic solution (solution B20) was developed for short periods of ischemia (2 to 3 hours) [Monique Bernard et al. J Thorac. Cadiovasc Surg 90: 235-242, 1985].
If the needs expressed by the surgeons are considered, only the option of an extension of the duration of preservation to 12 hours or 6 months or more presents any importance. A passage of 4 to 12 hours of preservation in effect corresponds much more to a marginal increase in the duration of ischemia. It presents several advantages; (1) an extension of the geographical area in which the collection of the transplanted organs takes place and thus an increase in the chances of transplantation for a given patient by offering him the possibility to use a removed transplant, from geographically further away, (2) to be able to envisage transplanting by HLA compatibility freeing the necessary time to carry out the best possible donor-recipient matching (such transplants have already shown much better results in terms of survival of the transplant in renal transplantation, than the simple ABO matching currently used for heart transplants), (3) finally, the passage of 4 to 12 hours would leave at least 1 hour for the global non invasive evaluation of the metabolic state of the transplant by magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) without breach of the chain of sterility inasmuch as the study can be carried out in the preservation bag. Moreover, to date, no known solution allows preservation of the heart for a period longer than 4 hours, and all the more so during a period of 12 hours.
One of the aims of the invention is to suggest a solution of improved quality, used to arrest the heart then preserve it for a duration longer than 4 hours, with a view to transplantation.
One of the other aspects of the invention is to suggest a solution for arrest and preservation of the organ, especially the heart, making it possible to maintain cellular and metabolic integrity of the organs after ischemia.
Another aspect of the invention is to suggest a solution for organ preservation, especially the heart, allowing post-ischemic functional recovery.
The invention concerns a solution for perfusion and/or preservation and/or re-perfusion during organ transplant, especially the heart, characterised in that it contains the following elements:
The solution developed within the framework of the present invention for the arrest and the preservation of hearts for long term ischemia is such that it induces cardiac arrest with a strong concentration of magnesium rather than potassium. It comprises moreover glutamate (metabolic substrate of ATP in anaerobic) and is characterised by a non zero concentration in Ca2+ and a concentration in extracellular type Na+. Different protective agents, (lactobionate, raffinose, glutathion -reduced form-, allopurinol and adenosine) have been added to this solution. As far as certain components (adenosine, butanedione-2,3-monoxime) are concerned, research on the optimal concentration has been carried out. Recent work has underlined the importance of endothelial dysfunction in the damage linked to the ischemia-re-perfusion sequence. Because of this fact, L-arginine was included, precursor of NO, in the preservation solution.
By solution for re-perfusion during organ transplantation, is designated a solution useable during the transplant of an organ, to make the transplanted organ go from an ischemic state to the cardioplegic state.
According to an advantageous method of implementation, the solution of the invention does not contain butanedione 2.3-monoxime.
According to an advantageous method of implementation, the invention solution contains at least one water resistant agent chosen from lactobionic acid, mannitol and raffinose.
By order of preference, the water resistant agent is chosen from: lactobionic acid, mannitol and raffinose.
According to another advantageous method of implementation, the invention solution contains at least one agent to trap free radicals, chosen notably from glutathion (reduced form), allopurinol, or mannitol.
According to another advantageous method of implementation, the invention solution contains the following elements:
According to another advantageous method of implementation, the invention solution contains the following elements:
In the solution of the invention, Na+ is in the form NaOH, K+ is in the form KCl and KH2PO4, favourably at the rate of 2 to about 3.5 mM KCl and about 2 to about 3.5 mM KH2PO4, Ca2+ is in the form of CaCl2, 2H2O and Mg2+ is in the form of MgCl2, 6H2O.
The invention solution is such that it allows the preservation of organs, especially the heart for a duration of at least 12 to 15 hours and particularly for at least 12 hours.
The invention equally concerns a solution as described above, to maintain the cellular and metabolic integrity of the organ after ischemia, this integrity can be detected by measuring the activity of at least one of the following enzymes: lactate dehydrogenase, creatine kinase and/or by measurement of at least one of the following metabolites: purines, nucleotides especially adenylic nucleotides, inosine monophosphate, adenosine triphosphate, amino acids, inorganic phosphate, lactate, phosphocreatine.
The invention equally concerns a solution such as is described above, to ensure functional post ischemic recovery, this functional recovery able to be measured by the measurement of at least one of the following hemodynanic parameters: coronary flow, the developed pressure, the cardiac frequency, and the diastolic pressure.,
The quality of preservation is measured by studying the functional and metabolic parameters as well as the measurement of cellular integrity.
The measured functional parameters are:
the developed pressure,
the diastolic pressure,
the cardiac frequency, these three parameters being measured thanks to a small balloon placed in the left ventricle on which the heart contracts; the small balloon is linked to an amplifier (Statham P23dB) which is connected to a recorder,
and the coronary flow (measurement of the discharge coming from the heart during a given time).
The functional parameters are measured during the control period and during the period of re-perfusion.
The metabolic parameters measured are:
inorganic phosphate,
adenosine triphosphate,
phosphocreatine, and
intracellular pH.
The acquisition of RMN spectrums of P-31 makes it possible to follow the levels of Pi (inorganic phosphate), ATP (adenosine triphosphate), PCr (phosphocreatine) as well as the intracellular pH (ATP and PCr=high energy phosphorous components). The spectrums are acquired throughout the entire duration of the experiment (control, ischemia, re-perfusion).
The adenylic nucleotides, inosine monophosphate and the purines are measured in the frozen hearts at the end of the experiment; this measurement is additional to the results obtained from the RMN spectrums of the P-31.
The amino acids are measured in the frozen hearts at the end of the experiments. They play an important role in the metabolism and maintenance of cardiac cellular function. They are depleted during ischemia and re-perfusion. The size of this depletion is a reflection of the deterioration of the heart.
The inorganic phosphate (Pi) and the purines are measured in the discharge coming from the heart and are thus only measured during the periods of perfusion (control and re-perfusion). The Pi and the purines are accumulated during ischemia and come out of the heart at the moment of re-perfusion, representing a loss of precursors for the synthesis of the high energy compounds.
The lactate in the discharges (control and re-perfusion) is evaluated as an indicator of the metabolism of energy in anaerobia.
Parameters reflecting cellular integrity.
The leakage of creatine kinase (CK) in the discharges indicates cellular damage.
The quantities of creatine kinase (CK) and lactate dehydrogenase (LDH) in the frozen hearts at the end of the experiment equally represent an indicator of cellular integrity.
The myocardiac water content equally reflects cellular integrity.
In order to highlight the importance presented by the invention solution, this has been compared, with the aid of a large number of parameters and in comparison to the protection provided, to reference solutions that arc the St Thomas solution and the UW solution, used in the preservation of cardiac transplants. A comparison with the Broussais and Celsior solutions, used in certain French transplant centres has equally been done. The results show that the invention solution permits greater protection than that given by the other solutions:
St Thomas, UW, Broussais, Celsior (cf.figures).
The invention solution can be used during all the phases of a transplant:
1) to stop the heart of the donor (cardioplegic solution),
2) to preserve the heart (hypothermic storage, transportation),
3) during re-implantation in the recipient.
The invention solutions can be prepared in the following manner: the different constituents arc dissolved and diluted in distilled water, the pH is adjusted to 7.4 with NaOH. The solution is filtered on 0.21 xcexcm. The apyrogenic and sterile solution is prepared and kept away from oxygen in the air.
(Figures, Text)
ATP=adenosine triphosphate
ADP=adenosine diphosphate
AMP=adenosine monophosphate
PCr=phosphocreatine
Pi=inorganic phosphate
LDH=lactate dehydrogenase
CK=creatine kinase
HPLC=high pressure liquid chromatography
MRS=magnetic resonance spectroscopy
EDP=diastolic pressure
dP=developed pressure
CF=coronary flow
RPP=xe2x80x9crate pressure productxe2x80x9d=cardiac frequencyxc3x97developed pressure
NAD=nicotinamide adenine dinucleotide
In the following, a solution conforming to the invention, useable for isolated hearts, presents the following composition:
KCl (2 mM), KH2PO4 (2 mM), CaCl2 (0.25 mM), MgCl2 (13 mM), NaOH (120 mM), arginine (2 mM), glutamate (20 mM), adenosine (0.5 mM), lactobionic acid (100 mM), raffinose (30 mM), glutathion (3 mM), allopurinol (1 mM), mannitol (30 mM).
A solution in accordance with the invention, useable for heterotopic transplantation, has the following composition:
KCl (2 mM), KH2PO4 (2 mM), CaCl2 (0.25 mM), MgCl2 (13 mM), NaOH (120 mM), arginine (3 mM), glutamate (20 mM), adenosine (0.5 mM), lactobionic acid (100 mM), raffinose (30 mM), glutathion (3 mM), allopurinol (1 mM), mannitol (30 mM).
A solution with a composition similar to that of the invention, not containing arginine, is named as xe2x80x9cCRMBMxe2x80x9d in the following and has the following composition:
KCl (2 mM), KH2PO4 (2 mM), CaCl2 (0.25 mM), MgCl2 (13 mM), NaOH (120 mM), glutamate (20 mM), adenosine (0.5 mM), lactobionic acid (100 mM), raffinose (30 mM), glutathion (3 mM), allopurinol (1 mM), mannitol (30 mM).